In this paper, we clarify the relationship between myoblast orientation and myogenesis by pattering myoblasts and analyzing gene expression of self-assembling muscle tissue over time. Previous studies have shown that it is possible to pattern muscle cells by culturing cells on chemically or physically patterned surfaces. We applied this technique to uncover the how the physical placement of cells and mechanobiological adhesive surface sensing influence intercellular communication and intracellular dynamics resulting in the different form and functionality of the final muscle tissue. By using microchannels with different widths, we controlled the cell fusion directions and successfully fabricated muscle tissue with different orientation distributions. Furthermore, we analyzed mRNA expression and protein synthesis known to play a key role in myogenesis and revealed that the maturation speed accelerates by patterning myoblasts and the optimal channel width was estimated about 100 μm.